In situ hybridisation/ Visualising mRNA distribution in tissues Flashcards
What is the purpose of in-situ hybridisation?
The most visual way of determining where and when a gene is expressed in an embryo or a specific tissue, is by detecting acummulation of the messenger RNA. This can be done by performing an in-situ hybridisation
What is the difference between traditional in situ and whole mount?
Unlike traditional in situ hybridization techniques, which require thin tissue sections whose images will need to be computationally reassembled, the whole-mount technique allows gene expression patterns to be assessed over the entire embryo or structure.
What are used to detect mRNAs?
Synthetically produced RNA probes, called riboprobes, are used to detect mRNAs by complementary binding.
What are riboprobes labelled with?
Riboprobes are labeled with special nucleotides containing “haptens,” such as dinitrophenol, biotin, or digoxygenin.
What are haptens?
Haptens are molecules that can elicit an immune response when attached to larger molecules, and are therefore targets for antibody binding. These detection antibodies are conjugated to enzymes, such as horseradish peroxidase or alkaline phosphatase, that catalyze chemical reactions where a fluorescent or colored dye can be deposited.
What is the first step in the procedure?
the identification of the target sequence in the model organism to be investigated. The target DNA sequence is then amplified by PCR using primers containing RNA polymerase initiation sequences. The amplified DNA template is now transcribed in vitro with hapten-labeled nucleotides. This hapten-labeled riboprobe is now ready for the hybridization step.
What is involved in the hybridisation step?
Embryos are prepared for hybridization via fixation with formaldehyde, a cross-linking reagent that stabilizes proteins and protects against RNases. After fixation, the formaldehyde is removed by washing the embryo several times with phosphate buffered saline containing a small amount of detergent.
To remove cellular lipids and facilitate probe penetration into the tissues, embryos are dehydrated in a graded series of methanol washes
How do you prepare embryos for hybridisation?
To prepare embryos for hybridization, they must be rehydrated by a graded series of methanol washes with progressively less methanol per wash. Embryos are then digested with a protease to facilitate diffusion of riboprobe into the tissues. The labeled riboprobe is added to the embryo and the hybridization is carried out.
What is the final step?
Post-hybridization washes are performed to remove nonspecific hybridizations..
RNases A an T1 are added to remove incompletely hybridized probes by digesting single-stranded RNA. The hybridized probes are detected with an antibody-enzyme conjugate that binds the hapten-labeled riboprobes.
enzymes like alkaline phosphatase are conjugated to hapten-specific antibodies, and are detected by adding enzyme substrates to elicit a color change. The reaction product forms a dark purple precipitate marking the location of the expressed mRNA
In situ hybridisation protocol
1) Fix embryos in PFA 4% over-night (O/N) at 4°C
2) Wash in PBS
3) Wash in Methanol- Keep at -20°C O/N. They can be stored pretty much
forever at this step, but make sure they spend at least one night at -20°C before
continuing with the protocol.
4) Wash 2 times 5min in PBS triton 0.1% (PBST)
5) Proteinase K treatment: only for embryos 24hpf and older
PK stock at 10mg/ml
24hpf: 1:1000 (1X) for 10 min
30hpf: 1X 20min
48hpf: 1X 30min
2. 5dpf: 1.5X 30/40min
3dpf: 2X 30/40min
6) Wash gently twice with PBST
7) Refix with PFA 4% 20min at RT
8) wash 5 times 5min in PBST
9) Rinse in Hyb+/PBST 1:1
Hyb+: 5XSSC, 50%formamide, 0.1%triton, 5mg/ml torula RNA, 50ug/ml heparin
(SSC 20X: NaCl 3M; NaCitrate 0.3M)
10) Prehybridise in Hyb+ for at least 1h at 68°C
* At this point we can store them at -20°C for some time.
11) Replace with diluted probe (usually by default I would do a 1:300 if it is a
strong probe, and 1:100 if I don’t know how will it work)
Incubate O/N at 68°C
12) Recover probe
13) Wash 4 times 20min with Hyb-
Hyb-: 5XSSC, 50%formamide,0.1%triton (at 68°C)
14) Rinse once with 2XSSC (at 68°C)
15) Wash twice 20min with 0.2XSSC (at 68°C)
16) Wash 4 times 15min with PBST (back at room temperature)
17) Block for at least 1h with MABlock (0.1M maleic acid pH7.8+5%BSA from
Roche)
18) Incubate with anti-DIG antibody (Roche) 1:6000 O/N at 4°C
* Embryos can stay in step 18 for several days at 4°C
19) Wash 4 times 15min with PBST
20) Prepare staining buffer:
0.1M Tris HCL pH9.5 (keep 1M stock)
50mM MgCl (keep 1M stock)
0.1M NaCl (keep 4M stock)
0.1% triton (keep 10%stock)
Wash 3 times 5min in staining buffer
21) Prepare staining solution:
1ul NBT
3.5ul BCIP
in 1ml of staining buffer
Incubate in the dark, without agitation, check every once in a while.
22) Stop reaction washing a few times with PBST
23) Refix with PFA 4% O/N at 4°C
24) Wash in PBS, 30%, 50% and 70% glycerol. Store in 70% glycerol.
what is the importance of DIG?
During in situ, we have A probe (single stranded RNA molecule) that is complementary to the mRNA we want to detect. To this probe, we attach a large molecule that you can recognise with an antibody = DIG. So, when the labelled probe has been bound to the messenger RNA in the tissue, you can wash away the unbound probe and add an antibody that recognised the DIG molecule and wash away any of the antibody that hasnt bound, and to this antibody is conjugacted an enzyme = AP (alkaline phosphatase). Thus can add a substrate for that AP, so the substrate is converted into a coloured and insoluble preciptate that appears purple in the embryo.
